2.12 Immunoblot analysis
Protein concentrations were determined using a BCA Kit (Protein Assay Kit, Beyotime Biotechnology, Shanghai, China). 30 μg of total protein was loaded and separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Germany). Membranes were blocked with 5% (w/v) milk (Bio-Rad) in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) at room temperature for 1 h. Each nitrocellulose membrane was incubated with primary antibodies to phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technology; Cat#4370; 1:1000 dilutions), p44/42 MAPK (Erk1/2) (Cell Signaling Technology; Cat#9102; 1:1000 dilutions), GAPDH (Cell Signaling Technology; Cat#2118; 1:1000 dilutions) at 4 °C overnight. After three washes with TBST, Immune-reactive bands were detected by incubating with horseradish peroxidase (HRP) conjugated secondary antibodies (Santa Cruz Biotechnology; Cat#sc-2004; 1:3000 dilutions) at room temperature for 1 h. Then protein bands were incubated using the EasySee western Blot Kit (TransGen Biotech, Beijing, China) and visualized using enhanced chemiluminescence (ECL) reagents (Bio-Rad, Hercules, CA). Densitometric analysis was performed using Image J software (NIH, USA). The immune-related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al. , 2018).